Membrane Phospholipids: The Key Regulator of Tissue Factor Encryption/Decryption

 描述（由申请方提供）：凝血级联反应由凝血因子VIIa（FVIIa）与其细胞表面受体组织因子（TF）结合引发。组织因子是止血所必需的，但TF的异常表达或激活导致血栓形成，这是急性心肌梗死、不稳定型心绞痛和缺血性卒中的促发事件。它还会导致炎症和癌症。因此，TF表达和活性的适当调节不仅对维持止血平衡至关重要，而且对一般健康也至关重要。有趣的是，细胞表面的大部分TF以隐蔽状态存在，即，没有或几乎没有凝血活性，尽管与FVIIa形成复合物。需要刺激物使隐蔽的TF变成促凝血活性形式。多种细胞改变将隐蔽TF转化为凝血活性TF。我们最近的研究表明，4-羟基壬烯醛（HNE），一个最丰富的和生物活性的物质产生的脂质过氧化，激活TF在单核细胞和内皮细胞。目前，还不清楚凝血活性TF与TF加密和解密中涉及的神秘形式或机制有何不同。目前尚不清楚，是否磷脂存在于细胞膜的外叶发挥关键作用，在保持TF的隐蔽状态。在所有提出的机制中，细胞活化后质膜外叶阴离子磷脂的外化似乎是隐蔽TF转化为活性TF的主要机制。然而，其他机制，如蛋白质二硫键异构酶（PDI）介导的巯基-二硫键交换反应，也可能在某些细胞类型的TF激活中发挥重要作用。负责维持TF在幼稚细胞中处于隐蔽状态的机制和负责响应于病理生理学相关刺激而暴露阴离子磷脂的分子途径尚不清楚。以下具体目标旨在填补这些关于细胞表面TF活性调节的知识空白。目标1：测试一种新的假设，即质膜外叶中的鞘磷脂含量高是维持TF在细胞表面处于隐蔽状态的原因。在此，我们还将测试鞘磷脂在质膜中的水解在TF解密中起关键作用;目的2：阐明参与HNE外化磷脂酰丝氨酸和TF激活的信号传导机制，并确定PDI介导的巯基-二硫键交换途径和脂筏完整性在此过程中的贡献。为了加强在细胞系统中进行的观察，并更好地了解细胞膜脂质如何影响TF活性，我们还将对掺入确定脂质体中的纯化TF进行额外的研究。从拟议的研究中获得的数据将提供新的见解了解TF活性的调节细胞表面。总体而言，从拟议的研究中获得的知识将有助于了解血栓性疾病的发病机制，并将有助于设计更好的治疗策略，血栓性和出血性疾病。

 DESCRIPTION (provided by applicant): The coagulation cascade is initiated by binding of coagulation factor VIIa (FVIIa) to its cell surface receptor, tissue factor (TF). Tissue factor is essential for hemostasis, but the aberrant expression or activation of TF leads to thrombosis, the precipitating event in acute myocardial infarction, unstable angina, and ischemic stroke. It also contributes to inflammation and cancer. Therefore, the proper regulation of TF expression and the activity is critical for not only to maintenance of the hemostatic balance, but also for health in general. Interestingly, the majority of TF on cell surfaces exists in a cryptic state, i.., with no or little coagulant activity, despite forming complex with FVIIa. A stimulus is required fo cryptic TF to become procoagulant active form. A variety of cellular alterations transforms cryptic TF to coagulant active TF. Our recent studies show that 4- hydoxynonenal (HNE), one of the most abundant and bioactive species produced by the lipid peroxidation, activates TF in monocytic and endothelial cells. At present, it is unclear how the coagulant active TF differs from the cryptic form or mechanics involved in TF encryption and decryption. It is unknown, at present, whether phospholipids present in the outer leaflet of plasma membrane play a critical role in maintaining TF in the cryptic state. Of all the proposed mechanisms, externalization of anionic phospholipids at the outer leaflet of plasma membrane following cell activation appears to be the main mechanism for transformation of the cryptic TF into active TF. However, other mechanisms, such as protein disulfide isomerase (PDI)-mediated thiol-disulfide exchange reactions, may also play an important role in TF activation in certain cell types. Mechanisms responsible for maintaining TF in a cryptic state in naive cells and molecular pathways responsible for exposure of anionic phospholipids in response to pathophysiologically relevant stimuli are unknown. The following specific aims are designed to fill these gaps of the knowledge on the regulation of TF activity at the cell surface. Aim 1: Test a novel hypothesis that high sphingomyelin content in the outer leaflet of the plasma membrane is responsible for maintaining TF in its cryptic state at the cell surface. Here, we will also test that the hydrolysi of sphingomyelin in the plasma membrane plays a key role in TF decryption; Aim 2: Elucidate signaling mechanism(s) involved in externalization of phosphatidylserine and TF activation by HNE, and ascertain contribution of PDI-mediated thiol-disulfide exchange pathways and lipid raft integrity in this process. To strengthen observations made in cell systems, and to have better understanding of how cell membrane lipids influence TF activity, we will also perform additional studies with purified TF incorporated into defined liposomes. The data obtained from the proposed studies will provide new insights into understanding of the regulation of TF activity on cell surfaces. Overall, the knowledge gained from the proposed studies will be helpful in understanding the pathogenesis of thrombotic disorders and will be useful in designing better treatment strategies for both thrombotic and hemorrhagic diseases.